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A. Liu et al.
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Figure 1. IMiDs increase megakaryocytic colony formation and induce self-renewal and expansion of hematopoietic progeni- tors. (A) Purified CD34+ cells were cultured with POM (pomalidomide) or DMSO as vehi- cle control using MegaCult-C assay to ana- lyze formation of megakaryocytic colonies (CFU-Mk). Data shown are mean ± SEM from triplicates; Student’s t-test was per- formed, P-values are two-sided, **P<0.001 (B) Proliferation profiles of CD34+ cells expanded in serum-free HPGM hematopoi- etic growth medium supplemented with 10 ng/mL TPO with or without 10 μM IMiDs (LEN, lenalidomide or POM). Values shown are mean ± SEM of 7 separate cultures. P- values were calculated via one-way ANOVA with Bonferroni post hoc test. (*P<0.05 compared to vehicle; **P<0.001 compared to vehicle). (C) CD34+ cells cultured as described above were further analyzed by flow cytometry using propidium iodide (PI) staining for apoptosis and cell cycle at days 7 and 14 of culture. Cell pellets were stained for 30 minutes with equal volumes of phosphate-buffered saline (PBS) contain- ing 0.1 mg/mL PI and 0.6% Nonidet P-40 and 2 mg/mL RNAse. Cell cycle analysis was performed on a Beckman Coulter CyAN 9-color High Speed Flowcytometer, and data were analyzed by using Summit 4.3 soft- ware (Dako¬Cytomation) (left). P-values were calculated via one-way ANOVA with Bonferroni corrections. Right side is statistic result of triplicates. (D) CD34+ cells were treated with DMSO (0.01%), LEN, or POM (1 mM) for 3 days. The cells gated with CD34+ and analyzed by flow cytometry using propidium iodide (PI) staining for cell cycle. The result shown here is representative of two independent experiment.
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IMiDs treatments (day 3: vehicle 37.9%, LEN 42.2%, POM 42.5% in CD34+ cells (Figure 1D).
IMiDs inhibit megakaryocytic differentiation by blocking endomitosis and maturation
To characterize the CD34+ cells that exhibit increased proliferation and decreased apoptosis in long-term cul- tures with IMiDs, we performed flow cytometry after 14 days of culture in the presence of IL-3/IL-6/SCF with or without TPO. Flow cytometry revealed a strong induction of early CD34+ cells (CD34+CD38-) after 14 days of treat- ment with POM compared to vehicle (Figure 2A). Among these early CD34+ cells, more than 40% were double pos- itive for myeloid and megakaryocytic markers, expressing CD33+ and CD41+, after treatment with POM. The devel- opment of the immature myeloid/megakaryocytic “hybrids” (CD34+/CD38–/CD41+/CD33+) was independ- ent of TPO (vehicle 7.4%, POM 42.6%, POM+TPO 46.7%). Interestingly, the mean fluorescence intensity
(MFI) of CD33 in the CD34+CD38- immature hybrid cells strongly increased when cultured with TPO (MFI: vehicle 3.3, POM 5.9, POM+TPO 14.4). Furthermore, in the pres- ence of POM, we were able to culture and expand CD34+ cells for up to 4 months (Figure 2B). Characterization of cells from long-term cultures by multicolor flow cytome- try revealed two cell populations. First, 6.6% of the cells were CD45+34+33+11b+41+61+. These “hybrid” cells main- tained CD34+ expression with concomitant expression of myeloid (CD33, CD11b) and megakaryocytic (CD41 and CD61) markers. Second, 86.6% of the cells exhibited a more mature phenotype with loss of CD34 expression, but maintained co-expression of myeloid and megakary- ocytic markers, CD45+34-33+11b+41+61+. Despite the cul- ture conditions favoring thrombopoiesis, long-term cul- tured hematopoietic cells notably still expressed the myeloid markers CD11b and CD33, suggesting that IMiDs induce myeloid development. To address whether IMiDs have a sustained effect on expansion and self-
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haematologica | 2018; 103(10)